CN109852564A - Bacillus amyloliquefaciens 327 and its application - Google Patents
Bacillus amyloliquefaciens 327 and its application Download PDFInfo
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Abstract
The invention discloses a kind of bacillus amyloliquefaciens 327, are deposited in China typical culture collection center, and deposit number is CCTCC NO:M2019045.The invention also discloses a kind of application of bacillus amyloliquefaciens 327 in prevention and treatment tobacco black shank.Bacillus amyloliquefaciens 327 of the present invention have the ability of apparent antagonism tobacco black shank bacterium and the ability of antagonism pepper anthracnose.
Description
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens 327 and its applications, belong to technical field of biological control.
Background technique
Tobacco black shank (Phytophthoranicotianae) is most destructive soil-borne disease in Guizhou Tobacco production
One of evil, nearly ten million yuan of the economic loss as caused by the disease, seriously affects the income of tobacco grower every year.At present in production
Chemical control is mostly used greatly, but primary medicament service life is longer, pathogen drug resistance increases year by year, and pesticide residue and environment are dirty
The negative effects such as dye also gradually increase.Therefore, carrying out tobacco diseases prevention and treatment using biological control is the hot fields studied at present.
Bacillus (Bacillus spp.) is most common microbe species in current biological control research, gemma bar
Bacterium has the prospect of industrialization, and mechanism of action multiplicity, production cost is low, and easy storage and transportation is not likely to produce drug resistance, to safety of human and livestock etc.
Advantage is the important directions of biological prevention and control agent exploitation.Native Bacillus separation strains excellent there are many character domestic and international at present
As living microorganism preparation viable commercial product application, development potentiality is huge.The BioYield of Gustafson company research and development is by solving
Bacillus amyloliquefaciens (B.amyloliquefaciens) FZB42 is mixed with bacillus subtilis (B.subtilis) GB122 to be made
Fungicide, be applied to the prevention and treatment of plurality of plant diseases.China is earliest by China Agricultural University's development by waxy gemma
Yield increasing fungus series of products made of the bacillus such as bacillus (B.cereus) and bacillus subtilis (B.subtilis) rice,
There is good volume increase, protection effect on wheat, vegetables and more industrial crops.
It is suitable for the biological prevention and control agent exploitation of Guizhou Tobacco balck shank disease control at present and good preparation can be used
Relatively fewer, many reports prove that the drug resistance of Guizhou Province tobacco producing region balck shank pathogen increases year by year, it is therefore necessary to grind
Better biological inoculum is made to control the generation of tobacco black shank.
In addition, pepper anthracnose is one of the Major Diseases in capsicum production, it can cause harm in capsicum entire breeding time, make
At very big loss.Guizhou Province be the Main Cultivation of China capsicum one of, the long-term continuous cropping of capsicum causes the morbidity face of anthracnose
Product constantly expands, and seriously affects farmers' income.Production at present is upper mainly to use chemical pesticide control disease, the drug resistance of pathogen
Increase year by year, field dosage is also increase accordingly, and easily causes pesticide residue and environmental pollution.Therefore, using biological control into
The prevention and treatment of row pepper diseases is the hot fields studied at present.
Summary of the invention
The technical problem to be solved by the present invention is a kind of bacillus amyloliquefaciens 327 and its application are provided, to the black shin of tobacco
Disease and pepper anthracnose have preferable control efficiency, with overcome the deficiencies in the prior art.
The technical scheme is that a bacillus amyloliquefaciens 327, the strain is in preservation on December 22 in 2018
In China typical culture collection center (address: Wuhan, China Wuhan University), deposit number is CCTCC NO:M2019045,
Title are as follows: bacillus amyloliquefaciens 327 (Bacillus amyloliquefaciens 327).
The present invention also provides a kind of application of bacillus amyloliquefaciens 327 in prevention and treatment tobacco black shank.
The present invention also provides a kind of application of bacillus amyloliquefaciens 327 in prevention and treatment tobacco bacterial wilt.
The present invention also provides a kind of application of bacillus amyloliquefaciens 327 in prevention and treatment pepper anthracnose
The beneficial effects of the present invention are: the present invention acquires Rhizosphere Soil from Guizhou Province, tobacco planting plot, Affected It In Qianxinan Anlong County
Earth therefrom screens isolated bacillus 327, has apparent antagonism tobacco black shank bacterium (Phytophthora
) and the ability of the ability of ralstonia solanacearum (Ralatonia solanacearum) and antagonism pepper anthracnose nicotianae.
It is bacillus amyloliquefaciens by morphology, physio-biochemical characteristics and 16S rDNA identified for genes 327
(B.amyloliquefaciens)。
Detailed description of the invention
Fig. 1 bacillus screens the antagonistic effect of tobacco black shank bacterium, and CK: tobacco black shank bacterium (Phytophthora
nicotianae);327 indicate the Bacillus strain with antagonistic effect screened;
327 biomembrane of Fig. 2 bacterial strain, protease, cellulase, thermophilic iron element and phosphate detection;
327 16S rDNA phylogenetic trees of Fig. 3 bacterial strain;
The identification of 327 antimicrobial spectrum of Fig. 4 bacterial strain.
Specific embodiment
With reference to the accompanying drawing and invention is described further in specific embodiment:
One, the separation and screening of bacillus amyloliquefaciens 327
1. materials and methods
1.1 material
Soil sample is collected in 2017-2018 respectively from the serious plot of Guizhou Province's Affected It In Qianxinan Anlong County tobacco black shank morbidity
Healthy cigarette strain Rhizosphere sampling is acquired, using 5 point samplings, same plot soil sample is mixed and calculates 1 part of soil sample.Soil is collected using paper bag
Sample, soil collection tool sterilize in advance, and edaphon is avoided to mix.
1.2 culture medium
In separation preservation and plate face-off experiment of the LB culture medium for bacillus;PDA culture medium is mainly used for plate
In face-off experiment;Protease detects culture medium, cellulase detection culture medium, thermophilic iron element detection culture medium, phosphate detection
Culture medium is for detecting whether bacillus generates protease, cellulase, thermophilic iron element and phosphate etc..
1.3 tobacco diseases bacterial strains
Strains tested tobacco black shank bacterium (Phytophthora nicotianae) is by agricultural college, Guizhou University plant pathology
Teaching and research room is learned to provide.
2. the separation and preservation of rhizosphere bacillus
Using dilution-plate method, by soil dilution liquid, water-bath 30min is non-to kill the overwhelming majority in 85 DEG C of water-bath
Spore bacteria.100 μ L dilutions are taken to apply LB plate, every concentration applies cultivates at plate 3,37 DEG C.After culture 1 day, picking is different
Single colonie, carry out tube simultaneously be stored in -80 DEG C of refrigerators.
3. tobacco black shank bacterium and the screening of ralstonia solanacearum antagonistic Bacillus
Using tobacco black shank bacterium as test plant pathogen, prevention and treatment tobacco black shank effect is screened by tablet face-off method
Apparent biocontrol bacterial strain sees whether to generate inhibition zone by primary dcreening operation and secondary screening, and it is antibacterial straight using crossing method to measure its
Diameter, then detect whether by specific plate the generation of biomembrane, protease, cellulase, thermophilic iron element, phosphate etc., choosing
1 plant of the preferable bacillus of antagonistic effect is selected, number is 327 (see Fig. 1 and Fig. 2).
Bacteriostasis rate (%)=[(control group colony diameter-processing group colony diameter)/processing group colony diameter] × 100 2,
The identification of bacillus amyloliquefaciens 327
1. biocontrol bacterial strain is identified
It is identified by the bacterial strain that morphology, physiological and biochemical property and 16S rDNA gene pairs screen.
1.1 colony morphological observation
In inoculating strain to the test tube containing 5mL LB culture solution, 37 DEG C are shaken training overnight, and thalline were collected by centrifugation, thallus nothing
Bacterium water cleans 3 times, is finally resuspended with sterile water and is diluted to concentration 1 × 10810 μ L bacterium solutions are added dropwise in CFU/mL on LB plate,
37 DEG C be incubated overnight after observe colonial morphology.
1.2 physiological and biochemical property
According to " primary Jie Shi systematic bacteriology handbook ", the physio-biochemical characteristics of bacterial strain are measured.
1.3 molecular biology identification
37 DEG C of water-bath 1h of 50mg/mL lysozyme handle thallus, are mentioned using Biomiga bacterial genomes DNA extraction kit
Take genomic DNA.Using genomic DNA as template, PCR amplification is carried out using 16S rDNA universal primer 27F/1492R, PCR is produced
The raw work in sea is served after object verifying is correct carries out PCR product sequencing.Sequencing result carries out BLAST in ncbi database and compares analysis,
And the Neighbor-Joining in Multiple Sequence Alignment and phylogenetic analysis software MEGA6 is carried out using CLUSTAL X software
Algorithm phylogenetic tree construction.
Through analyzing: 327 can form more complicated colony structure (Fig. 2) on LB culture medium.The Physiology and biochemistry of bacterial strain is special
Property be shown in Table 1, strain bacterium is G+, can decompose glucose, rhamnose and mannitol, generate catalase and nitrate reductase, methyl
Red colouring is positive, the features such as liquefiable gelatin.By 16S rDNA gene sequencing, the 16S rDNA of bacterial strain 327 is constructed
Phylogenetic trees, the results show that bacterial strain 327 and bacillus amyloliquefaciens (Bacillus amyloliquefaciens) gather one
A branch (Fig. 3).It is bacillus amyloliquefaciens by morphology, physio-biochemical characteristics and 16S rDNA identified for genes 327
(B.amyloliquefaciens)。
1 bacterial strain GUAL203 physiological and biochemical property of table
+: positive reaction;: negative reaction
2. biocontrol bacterial strain antimicrobial spectrum is identified
Bacterial strain 327 has spectrum antagonistic activity, to tobacco black shank bacterium, tobacco ralstonia solanacearum, tobacco brown spot pathogen, capsicum
A variety of disease fungus fungistatic effects such as anthrax bacteria and sorghum A. mali are preferably (see Fig. 4).Show this plant of bacterium to the above disease
There is potential biocontrol effect.
3. biocontrol bacterial strain antibacterial material is identified
Bacterial strain 327 can produce thermophilic iron element and phosphate, promote plant growth related to bacillus.Bacillus is being planted
Object root colonization simultaneously generates complicated biofilm structure, can effectively occupy root table Advantageous sites, limitation pathogen mycelia is invaded
Enter, and can induce plant and resistance is generated to pathogen, achievees the effect that disease-preventing and yield-increasing.Three, bacillus amyloliquefaciens 327 are being prevented and treated
Application in terms of plant disease
1. tobacco black shank prevents and treats field trial
Experimental cultivar: cloud and mist 87.Test site: Guizhou Anlong.Experimental field: fertility is uniform, plantation flue-cured tobaccos in 2017 and black
Shin disease morbidity plot.
Experimental design: it is handled with bacillus amyloliquefaciens 327, with 30% first frost hymexazol aqua (middle peasant Li Hua of medicament
(Tianjin) agricultural chemicals Co., Ltd) it is positive control, with greenery patches health, (greenery patches health is the green health of middle peasant (Beijing) Limited Liability public affairs
The probiotics for taking charge of research and development, to improve the effect of soil micro-ecosystem controlling disease and growth-promoting volume increase), any medicine is not carried out
Agent processing is blank control, totally 3 processing, 3 repetitions, 30 plants of cigarettes of every cell, all random district's groups arrangements of processing.At medicament
It manages concentration and dosage to illustrate with reference to medicament, pouring root is applied when tobacco seedlings are transplanted, and is applied 2 times;Bacillus concentration is 108CFU/mL,
50mL bacterium solution pouring root application, co-administer 3 times, from tobacco seedlings transplant when pouring root 1 time, after every 7-10d pouring root 1 time.Field cultivation pipe
Reason method is consistent with locality, does not apply other fungicide.
Field investigation: the field investigation time is chosen and carries out 3 times altogether after the onset of blank control starts to harvesting is preceding, and investigation is all
Cell cigarette strain balck shank disease incidence is handled, and is classified investigation disease severity.Tobacco black shank disease scale standard is according to GB/T
23222-2008 is carried out, and calculates the disease incidence and disease index of disease, and data use the new multipole of DPS data processing system Duncan
Poor method carries out variance analysis.The results are shown in Table 2.
The field plot preventive effect of 2 Anlong area tobacco black shank of table
It is found that bacillus has good preventive effect to tobacco black shank, preventive effect slightly drops table 2 compared with first frost hymexazol
It is low, but application times are 1 time more.First frost hymexazol is that have in current production for preventing and treating the primary medicament of tobacco black shank
Report shows that tobacco black shank bacterium develops drug resistance to it in production.Therefore, in conjunction with the excellent of chemical agent and biological bactericide
Disadvantage can attempt biocontrol bacterial strain and compound application with chemical pesticide, reach the dosage for reducing chemical pesticide.
2. tobacco bacterial wilt prevents and treats field trial
Experimental cultivar: cloud and mist 87.Test site: Guizhou Anlong.Experimental field: fertility is uniform, plantation flue-cured tobaccos in 2017 and blueness
Blight morbidity plot.
Experimental design: it is handled with bacillus amyloliquefaciens 327, with Zhongshengmycin, (Zhongshengmycin is the Chinese Academy of Agricultural Sciences's biological and ecological methods to prevent plant disease, pests, and erosion
A kind of novel agricultural antibiotic succeeded in developing is the antibiotic generated by the mutation of Strepiomyces lavendulae Hainan, belongs to N- glucosides
Class alkali water-soluble substance.The processing dosage form of the bacterium is a kind of protective fungicide that fungicidal spectrum is wider, has and tags, permeates work
With.Zhongshengmycin has very high activity to bacterial disease and some fungi the venereal disease evil of crops, while having certain
Production-increasing function.It is biological bactericide more commonly used in current production.It is obvious to prevent and treat Solanaceae bacterial wilt effect) it is positive control,
With Pseudomonas fluorescens (commercial reagents), (Pseudomonas fluorescence is a kind of widely distributed Gram-negative rod bacterium, ring
Border is adaptable, has the function of inhibiting a variety of pathogens, helping plant absorption nutrition, promote plant growth etc. beneficial, is anti-
Control the specific drug of the bacterial diseases such as bacterial wilt, soft rot, bacterial angular leaf spot, rot disease, bacterial wilt of ginger.The disadvantage is that shelf life ratio
Shorter, 6 months viable counts are reduced after general production and bacterium activity declines, and be will lead to drug effect and are greatly lowered, can not sterilize
Agent uses, but can be mixed into compost and play some fertilizer efficiency effects.Bacillus can produce degeneration-resistant gemma, as fruit product contains
Water can achieve the shelf life of 2-3 less than 5% at present, can also long-term preservation, be bacillus as mainstream biological agent
The main reason for), not carrying out any chemicals treatment is blank control, totally 3 processing, 3 repetitions, 30 plants of cigarettes of every cell, institute
There is the random district's groups arrangement of processing.Chemicals treatment concentration and dosage illustrate that pouring root is applied when tobacco seedlings are transplanted with reference to medicament, apply 2 times;
Bacillus concentration is 108CFU/mL, 50mL bacterium solution pouring root application, co-administer 3 times, from tobacco seedlings transplant when pouring root 1 time, after every
7-10d pouring root 1 time.Field planting management method is consistent with locality, does not apply other fungicide.
Field investigation: the field investigation time is chosen and carries out 3 times altogether after the onset of blank control starts to harvesting is preceding, and investigation is all
Cell cigarette strain bacterial wilt disease incidence is handled, and is classified investigation disease severity.Tobacco bacterial wilt disease scale standard is according to GB/T
23222-2008 is carried out, and calculates the disease incidence and disease index of disease, and data use the new multipole of DPS data processing system Duncan
Poor method carries out variance analysis.The results are shown in Table 3.
The field plot preventive effect of 3 Anlong area tobacco bacterial wilt of table
The experimental results showed that bacillus is preferable to the preventive effect of tobacco bacterial wilt, the Pseudomonas fluorescens than commercialization is imitated
Fruit is not bad, can develop as Biological Control of Tobacco Diseases medicament.
3. pepper anthracnose prevents and treats field trial
Experimental cultivar: Dang Wu.Test site: Guiyang Huaxi District.
Experimental design: being handled with bacillus amyloliquefaciens 327, (the green health of the middle peasant (north the green Kang Wei of biological prevention and control agent is commercialized
Capital) Bioisystech Co., Ltd) it is used as positive control, not carrying out any chemicals treatment is blank control, totally 4 processing, 3 times
It repeats, 30 plants of capsicums of every cell, all random district's groups arrangements of processing.Chemicals treatment concentration and dosage are with reference to medicament explanation, capsicum
Pouring root is applied when transplantation of seedlings, is applied 3 times;Bacillus concentration is 108The application of CFU/mL, 50mL bacterium solution pouring root, co-administers 3 times,
From pouring root 1 time when capsicum transplantation of seedlings, after every 7-10d pouring root 1 time.Field planting management method is consistent with locality, does not apply it
Its fungicide.
The field investigation time is chosen after the onset of blank control starts and carries out 2 times altogether, and all processing cell capsicum anthraxs are investigated
Sick disease incidence, and it is classified investigation disease severity.Pepper anthracnose disease scale standard according to GB/T17980.33-2000 into
Row, calculates the disease incidence and disease index of disease, and data carry out variance using DPS data processing system Duncan duncan's new multiple range method
Analysis.
Start to carry out disease survey when blank control cell pepper anthracnose takes place.Investigation on May 22nd, 2018
It 1st time, investigates 1 time every 7-10d, investigates 3 times altogether.The results are shown in Table 4.
Field plot preventive effect of 4 bacillus of table to pepper anthracnose
The experimental results showed that this bacillus has certain preventive effect to pepper anthracnose, imitated with the biological prevention and control agent of commercialization
Fruit is suitable.
Bacillus amyloliquefaciens can produce a variety of antibacterial materials and inhibit phytopathogen.The study found that bacillus is anti-
Fungi activity derives from Cyclic lipopeptide antibiotic, and antibacterial activity then may be due to polyketone substance and Ribosome biogenesis approach
The bacteriocin of generation.These substances can inhibit the growth of bacterium and the growth of hypha,hyphae and the sprouting of spore.But it is different
The genome difference of bacillus is larger, therefore the antibacterial substance that isolated bacillus of the invention generates also needs further
Research.
It is suitable for the biological prevention and control agent exploitation of Guizhou Tobacco balck shank disease control at present and good preparation can be used
Relatively fewer, many reports prove that the drug resistance of the Guizhou Province tobacco producing region disease pathogen increases year by year, therefore this research is sought
The antagonistic Bacillus (Bacillus spp.) under Guizhou ecological zone and cropping pattern is looked for, and continues research and development biology
Preparation reduces Pesticide use and reduces pesticide residue, provides Research foundation to Guizhou Tobacco green production.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (4)
1. a bacillus amyloliquefaciens 327 are deposited in China typical culture collection center, deposit number is CCTCC NO:
M2019045。
2. application of the bacillus amyloliquefaciens 327 described in claim 1 in prevention and treatment tobacco black shank.
3. application of the bacillus amyloliquefaciens 327 described in claim 1 in prevention and treatment tobacco bacterial wilt.
4. application of the bacillus amyloliquefaciens 327 described in claim 1 in prevention and treatment pepper anthracnose.
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CN110257286A (en) * | 2019-06-19 | 2019-09-20 | 贵州省烟草公司遵义市公司 | One plant of bacillus amyloliquefaciens that can inhibit disease fungus |
CN114395397A (en) * | 2022-01-27 | 2022-04-26 | 云南农业大学 | Microbial soil improvement microbial inoculum for preventing and treating tobacco blight and preparation method thereof |
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